The present invention relates generally to plant molecular biology. More specifically, it relates to nucleic acids and methods for modulating their expression in plants.
Cell division plays a crucial role during all phases of plant development. The continuation of organogenesis and growth responses to a changing environment requires precise spatial, temporal and developmental regulation of cell division activity in meristems (and in cells with the capability to form new meristems such as in lateral root formation). Such control of cell division is also important in organs themselves (i.e. separate from meristems per se), for example, in leaf expansion, secondary growth, and endoreduplication.
A complex network controls cell proliferation in eukaryotes. Regulatory pathways communicate environmental constraints, such as nutrient availability, mitogenic signals such as growth factors or hormones, or developmental cues such as the transition from vegetative to reproductive. Ultimately, these regulatory pathways control the timing, frequency (rate), plane and position of cell divisions.
The basic mechanism of cell cycle control is conserved among eukaryotes. A catalytic protein serine/threonine kinase and an activating cyclin subunit control progress through the cell cycle. The protein kinase is generally referred to as a cyclin-dependent-kinase (CDK), whose activity is modulated by phosphorylation and dephosphorylation events and by their association with regulatory subunits called cyclins. CDKs require association with cyclins for activation, and the timing of activation is largely dependent upon cyclin expression. CDKs are a family of serine/threonine protein kinases that regulate individual cell cycle transitions.
Eukaryote genomes typically encode multiple cyclin and CDK genes. In higher eukaryotes, different members of the CDK family act in different stages of the cell cycle. Cyclin genes are classified according to the timing of their appearance during the cell cycle. In addition to cyclin and CDK subunits, CDKs are often physically associated with other proteins that alter localization, substrate specificity, or activity. A few examples of such CDK interacting proteins are the CDK inhibitors, members of the Retinoblastoma-associated protein (Rb) family, and the Constitutive Kinase Subunit (CKS).
The protein kinase activity of the complex is regulated by feedback control at certain checkpoints. At such checkpoints the CDK activity becomes limiting for further progress. When the feedback control network senses the completion of a checkpoint, CDK is activated and the cell passes through to the next checkpoint. Changes in CDK activity are regulated at multiple levels, including reversible phosphorylation of the cell cycle factors, changes in subcellular localization of the complex, and the rates of synthesis and destruction of limiting components. P. W. Doerner, Cell Cycle Regulation in Plants, Plant Physiol. (1994) 106:823-827.
Plants have unique developmental features that distinguish them from other eukaryotes. Plant cells do not migrate, and thus only cell division, expansion and programmed cell death determine morphogenesis. Organs are formed throughout the entire life span of the plant from specialized regions called meristems. In addition, many differentiated cells have the potential to both dedifferentiate and to reenter the cell cycle. There are also numerous examples of plant cell types that undergo endoreduplication, a process involving nuclear multiplication without cytokinesis. The study of plant cell cycle control genes is expected to contribute to the understanding of these unique phenomena. O. Shaul et al., Regulation of Cell Division in Arabidopsis, Critical Reviews in Plant Sciences, 15(2):97-112 (1996).
There is evidence to suggest that cells must be dividing for transformation to occur. It has also been observed that dividing cells represent only a fraction of cells that transiently express a transgene. Furthermore, the presence of damaged DNA in non-plant systems (similar to DNA introduced by particle gun or other physical means) has been well documented to rapidly induce cell cycle arrest (W. Siede, Cell cycle arrest in response to DNA damage: lessons from yeast, Mutation Res. 337(2): 73-84). Therefore, to optimize transformation it would be desirable to provide a method for increasing the number of cells undergoing division.
Cell division in higher eukaryotes is controlled by two main checkpoints in the cell cycle that prevent the cell from entering either M- or S-phase of the cycle prematurely. Evidence from yeast and mammalian systems has repeatedly shown that over-expression of key cell cycle activating genes can either trigger cell division in non-dividing cells, or stimulate division in previously dividing cells (i.e. the duration of the cell cycle is decreased and cell size is reduced). Examples of genes whose over-expression has been shown to stimulate cell division include cyclins (see, e.g. Doerner, P. et al., Nature (1996) 380:520-423; Wang, T. C., et al., Nature (1994) 369:669-671; Quelle D. E., et al., Genes Dev. (1993) 7:1559-1571, E2F transcription factors (see, e.g. Johnson D. G. et al., Nature l (1993) 365:349-352; Lukas, J. et al., (1996) Mol. Cell. Biol. 16:1047-1057), cdc25 (see, e.g. Bell, M. H. et al., (1993) Plant Molecular Biology 23:445-451; Draetta, D. et al., (1996) BBA 1332:53-63), and mdm2 (see, e.g. Teoh, G. et al., (1997) Blood 90:1982-1992). Conversely, other gene products have been found to participate in negative regulation and/or checkpoint control, effectively blocking or retarding progression through the cell cycle. (see MacLachlan, T. K. et al., (1995) Critical Rev. Eukaroytic Gene Expression 5(2):127-156).
Current methods for genetic engineering in maize require a specific cell type as the recipient of new DNA. These cells are found in relatively undifferentiated, rapidly growing callus cells or on the scutellar surface of the immature embryo (which gives rise to callus). Irrespective of the delivery method currently used, DNA is introduced into literally thousands of cells, yet transformants are recovered at frequencies of 10xe2x88x925 relative to transiently-expressing cells. Exacerbating this problem, the trauma that accompanies DNA introduction directs recipient cells into cell cycle arrest and accumulating evidence suggests that many of these cells are directed into apoptosis or programmed cell death. (Bowen et al., Tucson International Mol. Biol. Meetings).
Over the period between 1950 and 1980, the increase in maize production worldwide outpaced both wheat and rice. Despite a temporary downswing in the early to mid-1980""s (due to both environmental and political factors) world maize production has risen steadily from around 145 million tons in 1950 to nearly 500 million tons by 1990. Increases in yield and harvested area have been the predominant contributors to enhanced world production; with yield playing the major role in industrialized countries and area expansion being most important in developing countries. Yet, over the next ten years it""s also predicted that meeting the demand for corn worldwide will require an additional 20% over current production (Dowswell, C. R., Paliwal, R. L., Cantrell, R. P. 1996. Maize in the Third World, Westview Press, Boulder, Colo.).
The components most often associated with maize productivity are grain yield or whole-plant harvest for animal feed (in the forms of silage, fodder, or stover). Thus the relative growth of the vegetative or reproductive organs might be preferred, depending on the ultimate use of the crop. Whether the whole plant or the ear are harvested, overall yield will depend strongly on vigor and growth rate. In modern maize hybrids, the impact of heterosis on overall plant vigor and yield has been unarguably demonstrated (Duvick, D. N. 1984. In: Genetic contributions to yield gains in five major crop plants. W. R. Fehr, ed. CSSA, Madison, Wis.). Corn breeders since the 1930""s have been selectively breeding by identifying inbreds that in combination produce hybrid vigor well beyond either parent. Surprisingly little is known about why hybrids are so much larger than their parent inbreds, although there are some interesting observations in the literature. In metabolic studies, heterosis (increases over either parent) has been observed for physiological traits such as P uptake by roots (Baliger and Barber, 1979; Nielsen and Barber, 1978), but for many enzymatic traits the hybrid is often intermediate to the inbred parents (Hageman, R. H., Leng, E. R., Dudley, J. W. 1967. Adv. Agron. 19:45-86; Chevalier, P., Schrader, L. E. 1977. Crop Sci. 17:897-901; Schrader, L. E. 1974. Crop Sci. 14:201-205; Schrader, L. E. 1985. PP 79-89. In: Exploitation of physiological and genetic variability to enhance crop productivity. Harper, J. E. ed. Am. Soc. Plant Physiol. Rockville, Md., Schrader, L. E., Cataldo, D. A., Peterson, D. M., Vogelzang, R. D. 1974. Plant Physiol. 32:337-341).
Anatomical data is less confusing. In summarizing data from an earlier publication, Kiesselbach states that approximately 10% of the increased vigor of the hybrid over its inbred parents is due to cell enlargement, and 90% can be accounted for simply by increased cell numbers (Kiesselbach, T. A. 1922, 1949. The Structure and Reproduction of Corn, Nebraska Agric. Exp. Stn. Res. Bull. 161.). This evidence for enhanced cell divisions contributing to increased maize vigor remains unchallenged. Recently it was shown that overexpressing a B cyclin in Arabidopsis resulted in increased root biomass and the root cells were smaller (indicative of accelerated cell division), but the overall plant morphology was not perturbed (Doerner et al., 1996). Similarly, expression of maize CycD genes in corn will enhance growth and biomass accumulation.
Other more specialized applications exist for these genes at the whole plant level. It has been demonstrated that endoreduplication occurs in numerous cell types within plants, but this is particularly prevalent in maize endosperm, the primary seed storage tissue. Under the direction of endosperm-specific promoters, expression of CycD genes (and possibly expression of CycD in conjunction with genes that inhibit mitosis) will further stimulate the process of endoreduplication.
Generally, it is the object of the present invention to provide nucleic acids and proteins relating to the control of cell division.
It is another object of the present invention to provide nucleic acids and proteins that can be used to identify other interacting proteins involved in cell cycle regulation.
It is another object of the present invention to provide antigenic fragments of the proteins of the present invention.
It is another object of the present invention to provide transgenic plants comprising the nucleic acids of the present invention.
It is another object of the present invention to provide methods for modulating, in a transgenic plant, the expression of the nucleic acids of the present invention.
It is another object of the present invention to provide a method for increasing the number of cells undergoing cell division.
It is another object of the present invention to provide a method for increasing crop yield.
It is another object of the present invention to provide a method for improving transformation frequencies.
It is another object of the present invention to provide a method for providing a positive growth advantage in a plant comprising modulating CycD protein expression.
It is another object of the present invention to provide a method for modulating cell growth.
It is another object of the present invention to provide a method for modulating cell division.
It is another object of the present invention to provide a method for modulating plant height or size.
It is another object of the present invention to provide a method for providing a positive growth advantage.
It is another object of the present invention to provide a method for increasing the growth rate.
It is another object of the present invention to provide a method for enhancing or inhibiting organ growth, for example seed, root, shoot, ear, tassel, stalk, pollen, stamen.
It is another object of the present invention to provide a method for producing organ ablation.
It is another object of the present invention to provide a method for producing parthenocarpic fruits.
It is another object of the present invention to provide a method for producing male sterile plants.
It is another object of the present invention to provide a method for enhancing embryogenic response, i.e. size or growth rate.
It is another object of the present invention to provide a method for increasing callus induction.
It is another object of the present invention to provide a method for positive selection.
It is another object of the present invention to provide a method for increasing plant regeneration.
It is another object of the present invention to provide a method for altering the percent of time that cells are arrested, i.e. in G1 or G0 stages of the cell cycle.
It is another object of the present invention to provide a method for altering the amount of time a cell spends in a particular cell cycle.
It is another object of the present invention to provide a method for improving in cells the response to environmental stress such as drought, heat, or cold.
It is another object of the present invention to provide a method for increasing the number of pods per plant.
It is another object of the present invention to provide a method for increasing the number of seeds per pod or ear.
It is another object of the present invention to provide a method for altering the lag time in seed development.
It is another object of the present invention to provide a method for providing hormone independent cell growth.
It is another object of the present invention to provide a method for increasing growth rate of cells in bioreactors.
Therefore, in one aspect, the present invention relates to an isolated nucleic acid comprising a member selected from the group consisting of:
(a) a polynucleotide that encodes a polypeptide of SEQ ID NOS: 1, 11, 13, or 21;
(b) a polynucleotide amplified from a monocot nucleic acid library using the primers of SEQ ID NOS: 3-10, 15-20 or 23-30;
(c) a polynucleotide having 20 contiguous bases of SEQ ID NOS: 1, 11, 13, or 21;
(d) a polynucleotide encoding a monocot cyclin D protein;
(e) a polynucleotide having at least 70% identity to the entire coding region of SEQ ID NOS: 1, 11, 13, or 21, wherein the % identity is determined by GCG/bestfit program using a gap creation penalty of 50 and a gap extension penalty of 3;
(f) a polynucleotide that hybridizes under stringent conditions to a nucleic acid characterized by SEQ ID NOS: 1, 11, 13, or 21, wherein the conditions include a wash in 0.1xc3x97SSC at 60 to 65xc2x0 C.;
(g) a polynucleotide characterized by the sequences set forth in SEQ ID NOS: 1, 11, 13, or 21;
(h) An isolated nucleic acid amplified from a Zea mays nucleic acid library using the primers of SEQ ID NOS: 3-10, 15-20 or 23-30;
(i) a polynucleotide complementary to a polynucleotide of (a) through (g); and
(j) a polynucleotide having the sequence of ATCC deposit having the Designation No. 98847 or 98848.
In another aspect, the present invention relates to recombinant expression cassettes, comprising the nucleic acid operably linked to a promoter.
In some embodiments, the nucleic acid is operably linked in antisense orientation to the promoter.
In another aspect, the present invention is directed to a host cell transfected with the recombinant expression cassette as described, supra.
In a further aspect, the present invention relates to an isolated protein comprising a polypeptide of at least 10 contiguous amino acids encoded by the isolated nucleic acid. In some embodiments, the polypeptide has a sequence selected from the group consisting of SEQ ID NOS: 2, 12, 14, and 22.
In another aspect, the present invention relates to an isolated nucleic acid comprising a polynucleotide of at least 25 nucleotides in length which selectively hybridizes under stringent conditions to a nucleic acid selected from the group consisting of SEQ ID NOS: 1, 11, 13, and 21, or a complement thereof. In some embodiments, the isolated nucleic acid is operably linked to a promoter.
In yet another aspect, the present invention relates to an isolated nucleic acid comprising a polynucleotide, the polynucleotide having at least 80% sequence identity to an identical length of a nucleic acid selected from the group consisting of SEQ ID NOS: 1, 11, 13, and 21 or a complement thereof.
In another aspect, the present invention relates to an isolated nucleic acid comprising a polynucleotide having a sequence of a nucleic acid amplified from a Zea mays nucleic acid library using the primers selected from the group consisting of SEQ ID NOS: 3-10, 15-20, and 23-30 or complements thereof. In some embodiments, the nucleic acid library is a cDNA library.
In another aspect, the present invention relates to a recombinant expression cassette comprising a nucleic acid amplified from a library as referred to supra, wherein the nucleic acid is operably linked to a promoter.
In some embodiments, the present invention relates to a host cell transfected with this recombinant expression cassette.
In some embodiments, the present invention relates to a protein of the present invention that is produced from this host cell.
In an additional aspect, the present invention is directed to an isolated nucleic acid comprising a polynucleotide encoding a polypeptide wherein: (a) the polypeptide comprises at least 10 contiguous amino acid residues from a first polypeptide selected from the group consisting of SEQ ID NOS: 2, 12, 14, and 22; (b) the polypeptide does not bind to antisera raised against the first polypeptide which has been fully immunosorbed with the first polypeptide; and (c) the polypeptide has a molecular weight in non-glycosylated form within 10% of the first polypeptide.
In a further aspect, the present invention relates to a heterologous promoter operably linked to a non-isolated polynucleotide of the present invention, wherein the polypeptide is encoded by a nucleic acid amplified from a nucleic acid library.
In yet another aspect, the present invention relates to a transgenic plant comprising a recombinant expression cassette comprising a plant promoter operably linked to any of the isolated nucleic acids of the present invention. The present invention also provides transgenic seed from the transgenic plant.
In a further aspect, the present invention relates to a method of modulating expression of the genes encoding the proteins of the present invention in a plant, comprising the steps of (a) transforming a plant cell with a recombinant expression cassette comprising a polynucleotide of the present invention operably linked to a promoter; (b) growing the plant cell under plant growing conditions; and (c) inducing expression of the polynucleotide for a time sufficient to modulate expression of the genes in the plant. Expression of the genes encoding the proteins of the present invention can be increased or decreased relative to a non-transformed control plant.
In another aspect of the invention an isolated protein is provided comprising a member selected from the group consisting of:
(a) a polypeptide comprising at least 25 contiguous amino acids of SEQ ID NOS: 2, 12, 14, or 22;
(b) a polypeptide which is a monocot cyclin D protein;
(c) a polypeptide comprising at least 65% sequence identity to SEQ ID NOS: 2, 12, 14, or 22, wherein the % sequence identity is based on the entire sequence and is determined by GAP 10 using default parameters;
(d) a polypeptide encoded by a nucleic acid of claim 1; and
(e) a polypeptide characterized by SEQ ID NO: 2, 12, 14, or 22.